The maintenance of adequate and balanced deoxyribonucleotide (dNTP) pools is essential for faithful DNA replication and repair. Loss of normal control of the dNTP pools can lead to cell death, genomic instability, and predisposition to cancer in humans. Regulation of ribonucleotide reductase (RNR) is largely responsible for controlling the relative ratios and amounts of the cellular dNTP pools. The central role of RNR in dNTP biosynthesis has also made it a successful target in the treatment of a number of malignancies. The RNR enzyme comprises of two subunits: the R1 subunit binds the four NDP substrates as well as the allosteric effectors (NTPs and dATP) that govern substrate specificity and turnover rate, and the R2 subunit houses the essential tyrosyl radical required to initiate nucleotide reduction in R1. The enzymatic activity of RNR can be modulated by allostery, transcription, protein inhibitor association, and subcellular compartmentation of its subunits. The budding yeast S. cerevisiae has emerged as a prototypical model system with which to investigate the complex mechanisms regulating the RNR activity. This proposal focuses on the mechanisms that control the RNR activity and consequently cellular dNTP pools by using a combination of biochemical, cell biology, and genetic approaches. Our central hypothesis is that these regulatory mechanisms are integrated to maintain optimal dNTP pools under different growth conditions so as to ensure high fidelity DNA synthesis and repair. Three specific aims are proposed: (1) To test the hypothesis that the Sml1 protein inhibits the RNR enzyme by impeding regeneration of the R1 active site and to define molecular determinants of the R1-Sml1 interaction and Sml1 degradation; (2) To examine the regulation of subcellular localization of the R2 subunit by the cell cycle and DNA damage checkpoints; (3) To characterize the role of the newly identified small protein Sld1 in RNR regulation. Sld1 belongs to a family of small protein RNR regulators, including the S. cerevisiae Sml1 and Sld1, the S. pombe Spd1, and their homologs encoded by other fungal genomes. The emphasis is to gain a mechanistic understanding of how the RNR activity is controlled by these evolutionarily conserved small size RNR regulatory proteins. Lessons learned from studies of the yeast RNR will serve as a paradigm for understanding of RNR regulation and dNTP pool control in eukaryotic cells, and may also suggest new approaches for RNR inhibition and for antitumor and antiviral drug development. PUBLIC HEALTH RELEVENCE: Ribonucleotide reductase is an essential enzyme that provides the building blocks for DNA in all organisms. This enzyme is also a proven target of clinical treatment of human cancers. The overall goal of this project is to understand how the activity of this enzyme is controlled inside the cell and finding new strategy for drug development targeting this enzyme.